Project Summary Understanding how a cell is switched off and maintains its quiescence is fundamentally as important as how it is activated. The long-term goal of the proposed research is to determine how cell-intrinsic processes control and modulate activation states of macrophages in vivo. Because dysregulation and unprovoked activation of the immune system cause a host of human diseases associated with inappropriate inflammation, deciphering the molecular networks regulating immune activation normally is critical for addressing these health challenges. This will lead to new knowledge and technologies needed to harness the properties of macrophages for disease prevention and treatment. We are leveraging the unique advantages of the highly tractable vertebrate model system, Danio rerio, for exquisite genetic manipulations, high throughput screening, and in vivo imaging to dissect the complex relationship between intrinsic metabolic signaling and macrophage activation. The proposal encompasses a series of projects that collectively define essential negative regulators and their functions for keeping the innate immune system in check to maintain a normal equilibrium in macrophages. The starting basis of our projects stems from emerging evidence that metabolic and immune signaling pathways intersect to shape immune activation in macrophages, and a discovery of a null mutation in an intracellular NOD-like receptor (NLR) in zebrafish. A gene inactivation in this novel NLR, nlrc3l, causes unprovoked macrophage activation possibly due to metabolic dysregulation. The proposal seeks to define the network of molecular interactions of nlrc3l to understand this very important mechanism that keeps macrophages in check under normal biological conditions. We are taking a highly integrated approach at multiple levels-- using differential transcriptomics, proteomics, and metabolomics to inform candidate genes and pathways that constitute possible interactors and effectors of nlrc3l, and validating interactions using genetic mutants and biochemical studies. The proposal will also use the power of a forward genetic screen to discover additional genes akin to nlrc3l that prevent macrophage activation at steady state that act in the same or completely new pathways. We designed an innovative assay for the screen to assess macrophage activation using a live-cell reporter for an activation marker irg1. Finally, the proposal will examine the influence of lipid and glucose metabolic pathways on macrophage activation in zebrafish using genetic analyses and chemical screening. This work will benefit from collaboration with an expert group in extending our findings to mouse and human models. Taken together, these projects provide the important foundation for understanding the genetic and metabolic basis of how the innate immune system is kept in check, and will impact the direction of my lab far beyond the 5 years of MIRA funding.